Novel Non-Toxic Larvicide

ABSTRACT

An larvicide comprising an essential oil encapsulated within a non-viable yeast cell. The larvicide is particularly effective against mosquito larvae, non-toxic to humans and other non-target species, inexpensive to make, and non-toxic during manufacture, transport, and storage.

CROSS-REFERENCE TO RELATED APPLICATIONS

The following application claims benefit of U.S. Provisional ApplicationNos. 62/148,774, filed Apr. 17, 2015, 62/289,394, filed Feb. 1, 2016,and 62/294,174, filed Feb. 11, 2016, each of which is herebyincorporated by reference in its entirety.

BACKGROUND

Various insects are known carriers for pathogens of human and/ornon-human disease and/or are linked to the destruction of crops and/orother undesired outcomes. Thus, significant resources are devoted tolimiting and/or controlling various “pest” insect populations. Forexample, mosquitos are known carriers for pathogens of diseasesincluding, but not limited to, malaria (Anopheles) Zika virus, denguevirus, yellow fever, (Aedes) and West Nile virus (Culex). Accordingly,it is very desirable to kill pest insects like mosquitos at the larvalstage, before they can spread disease and infection.

Unfortunately the most commonly used method for limiting and/orcontrolling undesirable insect populations are pesticides which areoften harmful to humans and other non-target species. In the case ofmosquitos and other water born pests, many communities resort to addingsynthetic pesticides to water reservoirs, including sources of potablewater, for mosquito control. The synthetic pesticides used areneurotoxins and growth inhibitors. Their dispersal in the water supplyposes a risk to these communities. Furthermore, the manufacture, storageand transport of chemical pesticides all present potential hazards tohumans, animals, and/or other non-target species.

Other methods for controlling insect populations, such as theengineering of genetically modified insects are expensive and currentlyavailable in only limited areas and only for a specific variety ofmosquito (Aedes). Furthermore, because it is not always possible tocontrol the movement or migration of an insect population, geneticmodification may not be a viable mechanism for populations that areconsidered pests in a particular region, but which are benign or evenbeneficial in other regions. Furthermore, because this technology is newand largely untested, it's difficult to predict the long-termconsequences and efficacy of releasing genetically modified populationsof mosquitos.

Accordingly, novel methods of controlling pest insect populations thatare non-toxic to humans, animals, and/or desirable insect populationsare thus desirable. However, while non-toxic (to human and otheranimals) substances such as essential oils have been shown to beeffective in killing insect larvae, deployment of essential oils to pestpopulations is problematic, as large amounts of essential oil would haveto be repeatedly added to oviposition sites to achieve significantreduction in the pest population. Furthermore, the dispersed oils wouldthen be vulnerable to degradation by UV radiation and would disrupt theaquatic environment, with the potential for adverse effects onnon-target species. Accordingly, an effective mechanism for deliveringsubstances like essential oils directly to the pest larvae population isgreatly desired.

It should thus be well understood that because insects are ubiquitous,often prevalent in poor and/or remote communities, and most negativelyimpact vulnerable populations, methods of controlling pest insects thatare inexpensive, easy to manufacture, transport, store, and deploy,would be of great benefit.

SUMMARY

The present disclosure provides a novel insect larvicide that isnon-toxic to humans and other non-target species, inexpensive to make,and non-toxic during manufacture, transport, and storage. No harmfulwaste products are generated during the manufacture of this larvicideand all of its components are generally regarded as safe. Moreover,larvicidal element is effective only when the larvicide is consumed bylarvae of the pest insect. The present disclosure also provides methodfor making and using the novel larvicide.

DETAILED DESCRIPTION

The present disclosure provides a novel insect larvicide capsule that isnon-toxic to humans and other non-target species, inexpensive to make,and non-toxic during manufacture, transport, and storage. Moreover,according to various embodiments, the larvicidal element is effectiveonly when the capsule is consumed by the target larvae. The presentdisclosure also provides methods for making and using the novellarvicide.

For the purposes of the present disclosure the target larvae or targetspecies refers to the intended target of the larvicide. While many ofthe specific embodiments provided herein refer to mosquito larvae as theintended target, it will be understood that larvae of other insects orother species may also be the intended target and that the larvicide maybe altered, as described herein, to be more particularly suited towardsone target or another. Furthermore, it will be understood that the novellarvicide described herein may be designed to be suitable for more thanone target and that references to “a” or “the” target species does notnecessarily preclude embodiments wherein there is more than one targetspecies.

According to various embodiments, the novel larvicide capsule comprisesa larvicidal element encapsulated in an ingestible delivery vehicle.According to various embodiments, the larvicidal element is a substancethat is non-toxic to humans and other non-target species, but whichnegatively impacts the ability of the target species to behave in anundesirable manner. For example, contact between the larvicidal elementand the target may result in the immediate or eventual death of thetarget. Alternatively, contact between the larvicidal element and thetarget may result in the larva being unable to transmit a diseasevector, sterile, or developmentally hindered in some other way.According to a specific embodiment, the larvicidal element is anessential oil.

Essential oils include terpene components and are naturally produced byplants to provide protection from larvae and adult insects, while beingnon-toxic to humans. For the purposes of the present disclosure,essential oils are defined as terpene containing oils produced byplants. For more than three decades, essential oils have been recognizedas cheap, effective larvicides. Essential oils are thought to exertlarvicidal effects through three different mechanisms: neurotoxicity,growth inhibition, and interruption of metabolic pathways. Thesimultaneous action of these mechanisms retards the evolution ofresistance to the larvicide. Examples of essential oils that aresuitable for use as larvicidal elements include, but are not necessarilylimited to lemongrass, thyme basil, cinnamon, peppermint, orange peel,and neem oils. Since the composition of essential oils varies, oils maybe combined to enhance larvicidal efficacy where the environment orlarval physiology provide opportunity. Suitable essential oils can bepurchased commercially at low cost or extracted from the plants fromwhich they are derived using standard techniques. As a specific exampleof an essential oil's known efficacy as a larvicide, lemongrass oil hasbeen shown in laboratory studies to achieve 100% larval killing withintwenty-four hours at concentrations of less than 50 ppm. Accordingly, ina more specific embodiment, the larvicidal element is or includeslemongrass oil.

For the purposes of the present disclosure the term “ingestible deliveryvehicle” is intended to mean an entity capable of encapsulating thelarvicidal element and generally sequestering it from the environmentuntil the delivery vehicle is ingested by the target species. Theingestible delivery vehicle is generally non-toxic to non-targetspecies. In general, the ingestible delivery vehicle should beattractive as a food source to the target species and have sufficientdurability in the environment in which it will encounter the targetspecies that it can withstand the conditions long enough to be ingestedby the target species. For example, many larvae are water-borne and/orfind nutrients in aquatic environments thus, in these circumstances theingestible delivery vehicle should not readily degrade in an aquaticenvironment. According to some embodiments the ingestible deliveryvehicle may be inert to all or most environments that do not replicatethe environmental conditions found in the digestive system of the targetspecies. Accordingly, to various embodiments, the ingestible deliveryvehicle may be an inactive or non-viable yeast cell. According to a morespecific embodiment, the ingestible delivery vehicle is a non-viableyeast cell of the S. cerevisae variety. It is a well-documented featureof larval biology that mosquito larvae will preferentially consume andreadily digest S. cerevisae. In fact, a recommended food for rearinglarvae in laboratory settings is S. cerevisae. Moreover, the cellmembrane of yeast cells is rich in beta-6-glucan, a polysaccharide, andchitin. Larvae have intestinal enzymes specialized for the digestion ofbeta-6-glucan to obtain chitin and beta glucans and are able to rapidlybreak down ingested yeast cell membranes. Other suitable ingestibledelivery vehicles may include (1) S. cerevisae genetically modified forgreater essential oil loading and a thicker cell membrane and (2) S.cerevisae opsonized with fragments of adult insect exoskeleton,bacteria, corn oil, corn sugar, and other phagostimulant elements of thelarval diet.

The larvicidal element may be encapsulated, infused, injected,entrapped, loaded, etc. (referred to herein collectively as“encapsulated” for ease of discussion) into the ingestible deliveryvehicle using any suitable method depending on the specific larvicidalelement and ingestible delivery vehicle being used. Examples of suitablemethods for encapsulating the larvicidal element in the ingestibledelivery vehicle include, but are not limited to, a combination of heatand agitation, plasmolyzation, and coacervation.

According to a specific embodiment wherein a larvicidal capsulecomprises an essential oil such as lemongrass oil as the larvicidalelement and a yeast cell such as an S. cerevisae cell as the ingestibledelivery vehicle, the lemongrass oil can be encapsulated within theyeast cell via a process using heat and agitation, as described ingreater detail in the Examples section below. The heat and agitationmethod results in the encapsulation of all components of the essentialoils without discrimination, including terpenes and aldehydes. However,molecules as large as 400,000 can freely diffuse through the cell wall.

Once the essential oil enters the cell, the yeast becomes nonviable andcannot replicate, thereby reducing or eliminating any potential impacton the environment during storage, transportation, and/or use. However,while the yeast cell is nonviable, the cell's thick outer membraneremains intact and thus sequesters the oil from the surroundingenvironment. In fact, after encapsulation, water/ethanol extraction isthe only non-enzymatic laboratory for removing the encapsulated oil. Asexplained above, some target species, such as mosquito larvae haveintestinal enzymes that are specialized for the digestion ofbeta-7-glucan, thus resulting in a system wherein the lemongrassoil/yeast cell capsule is essentially inert to all environments it islikely to encounter other than the specialized digestive systems of thetarget mosquito larvae. Furthermore, it should be noted that both yeastand lemongrass oil are commonly found in food and are entirely harmlessto humans.

According to a specific embodiment of use, the larvicidal capsules ofthe present disclosure could be distributed via (1) an air-waterdisplacement propulsion device to oviposition sites or (2) anauto-dissemination strategy using a cornstarch-based powdereddistributed at nesting sites. The larvae then consume the larvicidalcapsules and the yeast cell wall is broken down by enzymes in the gut of3^(rd) and 4^(th) larval instars, which liberates the essential oil(s)from the capsule, allowing the oil to act on the larvae, resulting inlarval death. In general this system could be used in additional to orinstead of existing municipal or rural larvicide/insecticide/other pestcontrol programs. Furthermore, because the presently described systemcan be used in environments where traditional chemical larvicides andinsecticides aren't used due to safety risks, the presently describedlarvicidal system can be used in high-value breeding sites, including indrinking water reservoirs and the like. Alternatively, as described ingreater detail below, the larvicidal capsule may be designed to piggyback female mosquitos, who then carry the capsules back to ovipositionsites.

Accordingly, the present disclosure provides methods for delivering ordirecting the larvicide towards or retaining the larvicide in specificdesired environments. For example, because the larvicide targets larvae,it may be desirable to direct and maintain the larvicide to ovipositionenvironments so as to ensure the larvae will have the opportunity toencounter and ingest the larvicide. According to some embodiments, thismay involve modifying the larvicidal capsule.

For example, as stated above, the larvicidal capsule may be incorporatedin a poweder to piggy back on female mosquitos, who can then carry thecapsules to known or unknown oviposition sites. For example, the A.Aegypti mosquito tends to rest in dry, sheltered areas such asresidential awnings and holes in trees, but also tend to visit manyoviposition sites. Accordingly, rather than trying to place thelarvicide at each oviposition site, it may be easier to place thelarvicide in known resting sites or areas that look like likely restingsites. The larvicidal capsules of the present disclosure may be coatedwith silica, cornstarch or another pH ˜7 soluble coating to produce apowder which can be spread at likely resting sites and which can then bepicked up and delivered to oviposition sites by female mosquitos.Moreover, anatomical difference between male and gravid femalemosquitoes could be exploited to improve targeting and transfer of thelarvicide to the oviposition sites. For example, the soluble coating maybe able to accommodate biofunctionalization for tuning adherence to andaquatic release from female mosquitoes. Soluble coatings may provideother advantages including increasing the effective lifespan of thelarvicide and or increasing the speed and efficacy of distribution.

As another example of possible larvicidal capsule modifications, thelarvicidal capsules of the present disclosure may be modified to achievecertain desired buoyancies. For example, mosquito larvae are know tohave different feeding behaviors, i.e. some are surface feeders whileothers are benthic (bottom) feeders. In order to ensure that thelarvicide reaches the different feeding populations, the presentdisclosure provides methods for producing capsules with differentbuoyancies, allowing the capsules to maintain different water levels, orto maintain the location of the capsules in, for example, moving orrunning water bodies.

According to an embodiment, the buoyancy of the capsule can be alteredby introducing air pockets in the capsule. For example, when theingestible delivery vehicle is a non-viable yeast cell, air pocketscould be introduced into the yeast membranes during the encapsulationstage via oxygen infusion. In general, by controlling the volume of theair pocket in relationship to the density of the contents of thecapsule, one can control the degree of buoyancy of the capsule, therebyproducing a capsule that would float on the surface of the water ormaintain a certain water depth.

Alternatively or additionally, the buoyancy of the capsule can bealtered by applying an adhesive element to the exterior of theingestible delivery vehicle. The presence of an adhesive elementpromotes clumping and facilitates sinking of the capsules. Suitableadhesive elements may take the form, for example of muco-adhesivecompounds such as doped alginates. These could be applied to theexterior of the capsules by painting, dipping, spraying, andimmersion/vacuum drying.

Combinations of air pockets and adhesive elements could be used to evenmore precisely fine tune the capsule so that it can maintain a desiredposition within the water column. Additionally, buoyancy of the capsulemay be altered by altering its essential oil loading capacity, e.g.through the use of plasmolyzers.

Alternative or additional modifications of the capsules include_opsonization with phagostimulants, membrane saturation withchemoattractants, and combination in biodynamic configurations tofacilitate larval feeding dynamics.

The terms and expressions that have been employed are used as terms ofdescription and not of limitation, and there is no intent in the use ofsuch terms and expressions to exclude any equivalent of the featuresshown and described or portions thereof, but it is recognized thatvarious modifications are possible within the scope of the invention asclaimed. Thus, it will be understood that although the present inventionhas been specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

EXAMPLES Encapsulation of Lemongrass Oil in S. Cerevisiae

S. cerevisae as fresh or dried baker's yeast is heated and proofed forviability.

Lemongrass oil, heated S. cerevisiae and distilled water are combined ina 1:2.5:4 volumetric ratio (Mixture 1).

Mixture 1 is agitated in a rotary incubator at 100 rpm and 40 C. for aminimum of 4 hours.

Mixture 1 is transferred to vials and centrifuged for 10 minutes at2000×g.

After decanting supernatant oil and water into beaker for lateranalysis, cells are washed 5 times with corn oil,

Cells are dried for 1 hour at 250 F. in a vented or vacuum drying oven.

Cells are washed 5× with corn oil.

Cells are dried for 2 hours at 250 F. in a vented or vacuum drying oven.

Cells not scheduled for immediate use may be freeze-dried for 48 hours.

Efficacy of Encapsulated Lemongrass Oil Larvicide Protocol

Third and fourth instar larvae are collected following a starvationperiod.

Experimental and control groups (25 larvae per group) allowed toaccommodate to 100 ml-200 ml distilled water in enamel bowls for 1 hour.

Experimental group fed essential oil micro-encapsulations, fourreplicates per concentration.

Test containers are maintained at 25=28 C., with 12/12 light/darkenvironment preferred.

Mortality after 24 and 48 hours with no additional nutrition

Determination of 50% and 90% mortality and inhibition of adult emergenceconcentrations.

Abbott's test: control vs. experimental mortality

What is claimed is:
 1. A larvicidal capsule comprising an essential oilencapsulated in an ingestible delivery vehicle.
 2. The larvicidalcapsule of claim 1 wherein the ingestible delivery vehicle is anon-viable yeast cell.
 3. The larvicidal capsule of claim 1 or 2 whereinthe essential oil is lemongrass oil.
 4. The larvicidal capsule of claim1 further comprising a mechanism for maintaining a desired positionwithin a body of water.
 5. The larvicidal capsule of claim 4 wherein themechanism for maintaining a desired position within a body of water is abuoyancy control mechanism.
 6. The larvicidal capsule of claim 5 whereinthe buoyancy control mechanism comprises an air pocket within thenon-viable yeast cell.
 7. The larvicidal capsule of claim 6 wherein theair pocket maintains the larvicidal capsule on the surface of the bodyof water.
 8. The larvicidal capsule of claim 6 wherein the air pocketmaintains the larvicidal capsule below the surface of the body of waterbut above the bottom of the body of water.
 9. The larvicidal capsule ofclaim 4 wherein the mechanism for maintaining a desired position withina body of water is an adhesive element that facilitates clumping ofmultiple larvicidal capsules.
 10. The larvicidal capsule of claim 9wherein the adhesive element is applied to the exterior of theingestible delivery vehicle.
 11. The larvicidal capsule of claim 6further comprising an adhesive element that facilitates clumping ofmultiple larvicidal capsules.
 12. The larvicidal capsule of claim 1further comprising a soluble coating.
 13. The larvicidal capsule ofclaim 12 wherein the soluble coating is silica.
 14. A method for forminga larvicidal capsule comprising encapsulating an essential oil within aningestible delivery vehicle.
 15. The method of claim 12 wherein theingestible delivery vehicle is a non-viable yeast cell.
 16. The methodof claim 14 or 15 wherein the essential oil is lemongrass oil.
 17. Themethod of claim 14 further comprising introducing a buoyancy controlmechanism into the larvicidal capsule.
 18. The method of claim 14further comprising coating the ingestible delivery vehicle with asoluble coating.
 19. A method for controlling a target pest populationcomprising; introducing to the target pest population a larvicidalcapsule comprising an essential oil encapsulated within an ingestibledelivery vehicle under suitable conditions that it is likely that larvaeof target pest population will ingest the larvicidal capsule.
 20. Themethod of claim 18 wherein introducing comprises positioning a powdercomprising the larvicidal capsules in an area wherein gravid adultfemale target pests are likely to congregate; wherein the powder adheresto the gravid female such that it is carried to oviposition sites.